Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer’s Patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood- and milk-borne retroviruses spanning three routes, was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue resident sentinel macrophages for establishing infection.

Information for data in Figure 4 Green-Gag_Blue-CD169_Red-CD11c.zip: Raw images of footpad cryosections from B6 mice 15 min after intrafootpad administration of MLV Gag-GFP virus particles (Green). Macrophages and DCs were identified using antibodies to CD169 (blue) and CD11c (red) respectively. Images taken using Leica DMi8 laser scanning microscope. z-planes taken 0.2 µm apart

Information for data in Figure 5 Mammary glands infection Gag-Green_Nuclei-blue.zip:  Raw immunostained images of cryosections obtained from mammary gland of a lactating dam infected as in A (7 dpi). MLV-infected epithelial cells (green) in the teats were identified using antibodies to MLV GlycoGag and nuclei were stained using Hoechst 3342 (blue). 3-D volume rendered images were generated from z-planes images taken 0.3 µm apart. Images taken using Nikon W1 spinning disk confocal microscope

Information for data in Figure 8_MLVgag-green_Actin-Red_CD169-Magenta.zip: Figure 8A : Raw images of cryosections from ligated gut tissue containing a Peyer’s Patch (PP) from B6 mice that were challenged for 1 h with MLV Gag-GFP (green) corresponding to 1-4 x 10⁵ IU based on comparative western blot analyses with antibodies to Gag with equivalent amounts of WT FrMLV.  The PP were fixed, embedded in OCT and frozen tissue sections were stained with phalloidin (red) to visualize actin in the PP tissue structure. Images were taken using Leica DMi8 laser scanning microscope. z-planes taken 0.2 µm apart. Figure 8A-2 another veiw: Same as 8A. The only difference was the position at which the images were acquired. The position was centered around a region where MLV gag-GFP particles was seen tranversing the intestinal epithelium. Figure 8B: Raw images of cryosections from PP. Macrophages identified using antibodies to CD169 (magenta) were predominantly located at the serosal side of PP. MLV Gag-GFP (green) viruses can be seen in close association with CD169⁺ macrophages.

Information for data in Figure 9_Neonatal peyers patch_ CD169 staining.zip: Raw images of a Peyer’s Patch cryosection from a 3-day old neonatal B6 mouse. Macrophages in the developing follicle were identified using antibodies to surface marker CD169 (red).

Related to Figure 4 in the manuscript: Sentinel macrophages mediate capture of incoming viruses at the injection site during subcutaneous challenge.

Related to Figure 5 in the manucript: Visualization of MLV transmission from mother to offspring.

Related to Figure 8 in the manucript: Gut-infiltrating MLV is captured by Peyer’s Patch-resident CD169⁺ macrophages

Related to Figure 9 in the manucript: CD169 contributes to retrovirus particle acquisition and establishment of infection during oral challenge